Recurrent oncogenic translocations involving Ig or TCR loci characterize human lymphoid malignancies. These translocations often derived from mis-repaired DNA double stand breaks (DSBs) generated during normal lymphocyte development. Availability of free DNA end, proper end-processing and the mis-joining of distal DNA ends form the three essential steps for chromosomal translocations. Developmental DSBs in lymphocytes are normally repaired by the non-homologous end-joining (NHEJ) pathway with help from the alternative-end joining (A-EJ) pathway(s) that preferentially use micro-homology (MH) at the junctions. NHEJ is also responsible for end-processing, such as opening the hairpin ends at Ig/ TCR loci generated during V(D) recombination. Sequence analyses revealed that NHEJ and A-EJ also mediate the mis-repairs that generate translocations. Thus understanding the mechanism and regulation of NHEJ and A-EJ have broad implications in lymphomagenesis. DNA-PKcs is the catalytic subunit of the DNA-dependent protein-kinase (DNA-PK), a NHEJ factor and a PI3K related protein kinase. In the absence of DNA-PKcs, direct end-ligation is largely normal, but hairpin opening-a form of end-processing required for sealed hairpin ends before ligation, is completely blocked. Here we report that in a knockin mouse model expressing the kinase-dead (KD) form of DNA-PKcs alone (DNA-PKcsKD/KD), direct end-ligation is completely blocked, leading to embryonic lethality similar to core NHEJ deficient (e.g.Lig4-/- ) mice. Co-deletion of Ku70 that is necessary for the recruitment of DNA-PKcs to DNA, rescues the embryonic development of DNA-PKcsKD/KD mice, indicating that DNA-PKcs protein regulates end-ligation at DNA ends. Despite end-ligation defects, DNA-PKcsKD/KD cells open hairpin normally, but only in the presence of normal ATM kinase activity, revealing a previous unrecognized role of ATM in hairpin opening. Finally in contrast to frequent A-EJ mediated IgH-Myc translocations and aggressive lymphomas in other NHEJ/p53 double deficient mice, lymphomas are rare in DNA-PKcsKD/KDp53-/- mice despite the severe genomic instability, indicating potentially defects in A-EJ. DNA-PKcs is auto-phosphorylated and phosphorylated by ATM upon DSBs. Based on these findings, we hypothesize that DNA-PKcs phosphorylation regulates hairpin opening (Aim1), end-ligation (Aim 2) and A-EJ (Aim3) to suppress lymphomagenesis. Aim1 will address how redundant phosphorylation of DNA-PKcs and Artemis by ATM and DNA-PKcs promotes hairpin opening. Aim 2 proposes to test the hypothesis that autophosphorylation of DNA- PKcs at the DNA ends is necessary for ends-ligation by identifying functional relevant autophosphorylation sites on DNA-PKcs. Aim 3 will test the hypotheses that DNA-PKcs KD protein physically blocks end-resection and suppress A-EJ and oncogenic translocation by characterizing class switch recombination (mediated by A- EJ and NHEJ) and IgH-myc oncogenic translocations in DNA-PKcsKD/KD cells and mice. Together these studies will determine the role of DNA-PKcs phosphorylation in DNA repair and translocations. In the future, DNA- PKcs phosphorylation will provide an attractive target to regulate NHEJ and A-EJ for cancer treatments.